Methods of neural conversion of human embryonic stem cells

• US 8,642,334 B2
• Filed: 02/17/2010
• Issued: 02/04/2014
• Est. Priority Date: 02/17/2009
• Status: Active Grant

• Current Assignee: Memorial Sloan-Kettering Cancer Center
• Original Assignee: Memorial Sloan-Kettering Cancer Center
• Inventors: Chambers, Stuart, Studer, Lorenz
• Primary Examiner(s): BERTOGLIO, VALARIE E
• Application Number:
  US13/201,137
• Publication Number:
  US 20120094381A1
• Time in Patent Office: 1,448 Days
• Field of Search: None
• US Class Current: 435/377
• CPC Class Codes: C12N 2500/90 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 2501/15 Transforming growth factor beta (TGF-β)
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• CPC Class Codes: C12N 2501/41 Hedgehog proteins; Cyclopamine (inhibitor)
• CPC Class Codes: C12N 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2501/60 Transcription factors
• CPC Class Codes: C12N 2501/998 Proteins not provided for elsewhere
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2506/45 from artificially induced pluripotent stem cells
• CPC Class Codes: C12N 5/0619 Neurons

Abstract

The present invention relates generally to the field of cell biology of stem cells, more specifically the directed differentiation of pluripotent or multipotent stem cells, including human embryonic stem cells (hESC), somatic stem cells, and induced human pluripotent stem cells (hiPSC) using novel culture conditions. Specifically, methods are provided for obtaining neural tissue, floor plate cells, and placode including induction of neural plate development in hESCs for obtaining midbrain dopamine (DA) neurons, motorneurons, and sensory neurons. Further, neural plate tissue obtained using methods of the present inventions are contemplated for use in co-cultures with other tissues as inducers for shifting differentiation pathways, i.e. patterning.

Claims

1. A method for inducing differentiation in stem cells, comprising,

a) providing:

i) a cell culture comprising human pluripotent stem cells,

ii) a first inhibitor of Small Mothers Against Decapentaplegic (SMAD) protein signaling, wherein said first inhibitor is selected from the group consisting of Noggin, a disulfide-linked homodimer of Noggin, Dorsomorphin, LDN-193189, and mixtures thereof, and

iii) a second inhibitor of Small Mothers Against Decapentaplegic (SMAD) protein signaling, wherein said second inhibitor is 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl] benzamide (SB431542), and

b) plating said stem cells in a culture medium,

c) contacting said plated pluripotent stem cells within 24-36 hours of said plating with said first inhibitor of Small Mothers Against Decapentaplegic (SMAD) protein signaling and said second inhibitor of Small Mothers Against Decapentaplegic (SMAD) protein signaling, and

d) inducing differentiation of said contacted pluripotent stem cells into a population of cultured neuroectodermal precursor cells.

2. The method of claim 1, wherein said method further comprises a step of removing said first inhibitor from said contacted pluripotent stem cells within 48 hours after contacting said contacted pluripotent stem cells with said first inhibitor wherein said population of cultured neuroectodeinial precursor cells comprise placode precursor cells.

3. The method of claim 1, wherein said neuroectodermal precursor cells are at least 10% up to 100% of said population of contacted pluripotent stem cells.

4. The method of claim 1, wherein said neuroectodermal precursor cells expresse a protein selected from the group consisting of a paired box gene 6 protein and a Six1 protein expressed in at least 20% of said population of neuroectodermal precursor cells.

5. The method of claim 1, wherein said pluripotent stem cells are selected from the group consisting of human embryonic stern cells (hESC), human somatic stem cells, and induced human pluripotent stem cells (hiPSC).

6. The method of claim 2, wherein said placode precursor cells further differentiate into Brn3a+progenitor cells under serum free conditions.

7. The method of claim 1, wherein said population of neuroectodermal precursor cells further differentiate into cells selected from the group consisting of central nervous system (CNS) progenitor cells, pattemable neuronal cells, dopamine positive neurons and motoneurons.

8. The method of claim 1, further comprising a step of contacting said plated pluripotent stem cells with, a modified recombinant protein at least 99% identical to a mouse Sonic Hedgehog N-terminal fragment within 60 hours of said contacting of said first inhibitor and said second inhibitor, wherein said population of neuroectodermal precursor cells further differentiate into a posterior floor plate tissue or floor plate cells.